Inulin is a simple, inert polysaccharide comprising a family of linear β-D-(2→1) polyfructofuranosyl α-D-glucoses, in which an unbranched chain of up to 100 fructose moieties or more is linked to a single terminal glucose, the end fructose-glucose pair incidentally being identical to sucrose. Inulin preparations therefore comprise neutral polysaccharides of simple, known composition, but which are molecularly polydisperse, with molecular weights ranging up to 16,000 or beyond. Inulin is the storage carbohydrate of Compositae and is cheaply available from dahlia tubers. It has a relatively hydrophobic, polyoxyethylene-like backbone, and this unusual structure plus its non-ionised nature allows recrystallisation and easy preparation in a very pure state.
Although the molecular composition of inulin is well known, the reported determinations of its solubility are conflicting. For example, the Merck Index (Thirteen Edition, 2001) describes inulin as “slightly soluble in cold water and organic solvents, soluble in hot”, whereas a quantitative study (Phelps, C. F., (1965) Biochem. J, 95:41-47) suggested that two distinct forms of inulin exist—the first obtained by precipitation from water, the second by precipitation from ethanol—both of which are substantially soluble in water at 37° C. It is also known that suspensions of inulin become less soluble on standing. The form obtained by precipitation from water is referred to as alpha-inulin (aIN), and the form obtained by precipitation from ethanol is known as beta inulin (bIN).
A third polymorphic form of particulate inulin, designated gamma inulin (gIN), is disclosed in U.S. Pat. Nos. 4,954,622 and 5,051,408, the contents of which are incorporated herein by reference. See also Cooper, P. D. and Carter, M., (1986) Molec. Immunol. 23(8):895-901, and Cooper, P. D. and Steele, E. J., (1988) Immunol. Cell Biol. 66:345-352.
This third polymorphic form is virtually insoluble in water at 37° C., but is soluble in concentrated solution (for example 50 mg/ml) only at temperatures in the range of 70° C.—80° C., as are the alpha and beta forms. This series of three polymorphic forms in which inulin crystallises may be characterised by their different solubility rates in aqueous media ranging from one instantly soluble at 23° C. (beta230 inulin) through a form soluble at 37° C. with a half-time of 8 minutes (alpha378 inulin) to a form virtually insoluble at 37° C. (gamma inulin). All forms are interconvertible, the more soluble and unstable progressing on standing to less soluble and more stable forms, only reversible by complete solution followed by recrystallisation, with the end product being the stable gIN.
Subsequently, it was disclosed that the activity of the gamma polymorphic form of inulin as an immunoactive agent, particularly an adjuvant, could be enhanced if particles of gIN were associated with an antigen-binding carrier material, and that this association provides synergistic effects. Thus, particles can be formed by association of gIN with aluminium hydroxide (alum) gel to form a gIN/alum hybrid preparation referred to as “Algammulin”—see also U.S. Pat. No. 5,476,844, the contents of which are incorporated herein by reference, and Cooper, P. D. and Steele, E. J., (1991) Vaccine 9:351-357.
Notwithstanding the preference stated in the above US patents for particles <1 μm in diameter, more recent and more accurate determinations of the size of the particles in the inulin preparations described in those earlier patents has revealed that, when measured in hydrated form, the finest of these earlier preparations turned out to have only a minority of particles <1 μm in diameter. This is the case even after treatment with an ultrasonic disruption device as described in U.S. Pat. No. 5,476,844.
However, the preparation of gIN and gIN/alum hybrid (Algammulin) by use of dIN in accordance with the present invention does result in ultra-fine formulations in which at least a majority of the particles have a diameter of <1 μm, as shown in the examples below. Thus the present invention achieves a particle size that the earlier patents set out, unsuccessfully, to achieve.
The ability to produce fine or ultrafine particles of inulin of less than 1 μm in diameter has therapeutic significance, with such particles being useful for example as adjuvants. In particular, the preparation of such particles is important in enhancing their biological activity and in reducing undesirable side effects such as local reactogenicity when the particles are used in human or non-human animal patients.
The present invention is therefore predicated on the serendipitous twofold observations of the very small but consistent residual turbidity after attempts to dissolve gIN particles above 50° C., resulting in the discovery of a novel polymorphic form of particulate inulin designated dIN, and the anomalous appearance and behaviour of these particles near their temperature of complete dissolution, resulting in the novel concept of fragmentation of such particles to form micronuclei for recrystallisation in fine or ultra-fine particulate form.
The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in Australia.